Messenger RNA (mRNA) profiling is an established but specialised forensic technique for confirmatory body fluid identification. It is particularly useful in cases where questioned fluid types lack reliable conventional tests or where greater specificity is required. Samples submitted for analysis are frequently mixtures of multiple body fluids from different contributors. Among other techniques, mRNA profiles may be generated using end-point reverse-transcription PCR (RT-PCR) or quantitative reverse-transcription PCR (RT-qPCR). RT-qPCR offers several advantages over end-point, including a wider dynamic range and the ability to quantify RNA, enabling more informative and potentially more robust interpretation of complex samples. To our knowledge, only end-point RT-PCR is currently used for mRNA profiling in casework.

In 2011, PHF Science (formerly ESR) were first to implement mRNA profiling in casework. The original assay, CellTyper, later evolved to CellTyper 2, both endpoint RT-PCR-based, detect peripheral blood, saliva, menstrual fluid, vaginal material, spermatozoa, and seminal fluid. We have since built on this, developing RT-qPCR multiplex assays which detect the same number of body fluids with greater performance (1). We are now on a journey to be the first laboratory to validate and implement RT-qPCR body fluid assays into casework.

Recently we have incorporated markers for rectal mucosa, nasal mucosa (2), and an additional marker for menstrual fluid. Twenty assays were evaluated across 70 samples and recursive feature elimination was used to support marker selection and optimise final performance. This expanded the range of detectable body fluids and improves discrimination between biologically similar sample types. In parallel, we are developing probabilistic (rather than categorical) interpretation strategies to incorporate biological and technical uncertainty to support casework reporting.

We will describe the development and evaluation of our expanded RT-qPCR body fluid identification assay, with parallel advances in probabilistic interpretation to improve sensitivity, efficiency, and robustness. We will highlight the advantages of RT-qPCR and compare it with the limitations of RT-PCR that have motivated this transition. Key challenges encountered during assay development will be discussed, along with proposed solutions and considerations for future directions. Together, these developments represent an important step toward more robust and informative body fluid identification.

Authors

• Courtney Lynch (The University of Auckland, New Zealand)
• Jessica Lowe (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Anna Lemalu (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Keshni Rasanayagam (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Johanna Veth (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Jayshree Patel (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Tiffany Anns (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Rebecca Richards (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• James Curran (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• SallyAnn Harbison (The New Zealand Institute for Public Health and Forensic Science, New Zealand)
• Rachel Fleming (The New Zealand Institute for Public Health and Forensic Science, New Zealand)

References

[1] Lynch, C. & Fleming, R. Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification. Science and Justice 63, 724–735 (2023).
[2] Chirnside, O., Lemalu, A. & Fleming, R. Identification of nasal mucosa markers for forensic mRNA body fluid determination. Forensic Sci. Int. Genet. 48, 102317 (2020).